The present invention relates to synthetic tripeptidic substrates to be used as reagents for the quantitative determination of plasminogen activators and inhibitors thereof, plasminogen preactivators, trypsin, trypsin inhibitors and trypsinogen in human and mammal body fluids and tissue extracts by photometric, spectrophotometric and fluorescence-photometric methods.
The human organism produces several activators which cause the conversion of the proenzyme plasminogen into the active lysis enzyme plasmin. This group of activators includes e.g. tissue and blood kinase and urokinase. These activators play an important role in the mechanism of blood coagulation. If these activators are produced in an abnormally high amount, there exists the danger of an increased fibrinolytic activity and hence of an increased bleeding tendency or hemorrhage. On the other hand, too small a production of these activators causes a disturbance of the equilibrium between coagulation capacity and fibrinolysis and hence an increased danger of thrombosis. The determination of plasminogen activators in body fluids and tissue extracts is, therefore, of great importance in the clinical practice as described e.g. by I. WITT in "Biochemie der Blutgerinnung und Fibrinolyse", Verlag Chemie, Weinheim, 1975, p. 119: "The determination of the fibrinolytical activity in plasma or serum serves in the first place to recognize hyperfibrinolytic conditions which accompany various diseases. Moreover, good results were often obtained over the past years in the lysis of intravascular thrombi by the administration of plasminogen activators. Besides, measurements of the fibrinolytic activity are also imperious for checking this thrombolytic therapy.". F. E. SMYRNIOTIS et al. [Thromb. Diath. et Haemorrh., vol. III, 257-70 (1959)] report a.o. the following: "In normal persons the production of urokinase is independent of age, sex and amount of urine. The urokinase production is increased after the occurrence of a myocardium infarction and after an attack of coronary insufficiency. The production is decreased in patients suffering from carcinosis, cardial blocking and uremia. These differences suggest that significant alterations in the fibrinolytic system of the plasma may occur as a result of these diseases.".
Up to now there exist no really reliable methods for the determination of plasminogen activators in body fluids and organ extracts. Principally, three methods are known:
1. Spontaneous lysis of a blood clot. Blood is allowed to clot either spontaneously or by the addition of thrombin, and the spontaneous lysis of the clot is observed at 37.degree. C. Lysis normally takes place only after 24 hours. This method is unspecific since the activator is not measured directly but via the lysis enzyme plasmin (cf. I. WITT, "Biochemie der Blutgerinnung und Fibrinolyse", Verlag Chemie, Weinheim, 1975, page 119).
2. Hydrolysis of casein. This method takes advantage of the property of plasmin to hydrolytically degrade casein. Casein is incubated with the sample to be tested. At the beginning and the end of the test trichloroacetic acid is added to an aliquot portion of the incubation mixture. The tyrosin content of the supernatant phase is determined by spectrophotometric measurement at 280 nm. From the tyrosin content thus determined the activator activity can be calculated approximately [cf. L. F. REMMERT et al., J. Biol. Chem. 181, 431 (1949)].
3. Esterolytic method (for urokinase). This method takes advantage of the property of urokinase to catalyse directly the hydrolysis of N.sup..alpha. -acetyl-L-lysine methyl ester. This method was also used for establishing the so-called CTA urokinase unit (CTA=Committee on Thrombolytic Agents of the National Heart Institute, USA). One CTA unit is the quantity of urokinase which releases 46.2.times.10.sup.-3 .mu.moles of methanol from N.sup..alpha. -acetyl-L-lysine methyl ester within 1 hour at 37.degree. C. (cf. N. U. BANG et al., "Thrombosis and Bleeding Disorders", Georg Thieme Verlag, Stuttgart, 1971, p. 377). This method can be applied exclusively to the determination of pure urokinase preparations but is not suited for the determination of urokinase in body fluids or tissue extracts since the said ester is split faster by proteolytic enzymes present therein (e.g. plasmin, plasma kallikrein, etc.) than by urokinase.
Attempts have been made to synthesize amide and peptide substrates for the determination of urokinase. However, these attempts were unsuccessful [cf. e.g., W. TROLL et al., Journal of Biological Chemistry 208, 85 (1954)].
In attempts to develop a synthetic substrate for the determination of kallikrein, the following tripeptide-p-nitroanilides ##STR2## were synthesized on the grounds of theoretical considerations. These tripeptides contain the last three C-terminal amino acids of the split products formed by the action of kallikrein on kininogen. Hence, it might have been expected that these two substrates would be hydrolyzed by kallikrein which, however, was true for substrate A only. On the other hand, it was quite unexpected to find that substrate B, which is not split by kallikrein at all, is rapidly amidolytically split by urokinase and other plasminogen activators. On the grounds of these findings a new class of tripeptidic substrates was developed.